A non-linear regression analysis method for quantitative resolution of the stimulus-evoked compound action potential from rodent optic nerve

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Abstract

The stimulus-evoked compound action potential (CAP) recorded from rodent optic nerve is polyphasic in profile, with total area under the CAP an index of nerve function. A decrease in CAP area signifies a decrease in the number of axons contributing to the CAP. A disadvantage of considering the CAP a uniform entity is that any distinctions between the axon-subpopulations contributing to the individual peaks go undetected. We illustrate two instances that demonstrate the advantages of resolving the CAP into its constituent peaks. The individual peaks of the CAP were quantified using curve-fitting procedures to describe the CAP as the sum of multiple Gaussian functions. The first example illustrates that the individual peaks comprising the CAP exhibit differential sensitivities to glucopenia, suggesting that the axons within the nerve cannot be considered a homogeneous population with regard to their metabolic characteristics. The second example illustrates that the complex waveform of the CAP recorded from the optic nerve of the hypomyelinated rumpshaker mutant mouse, comprising both positive and negative overlapping peaks, can be resolved into five individual peaks. In conclusion we show that assessing each individual peak of the CAP is a powerful analytic tool for identifying heterogeneous profiles of axon-subpopulations in the rodent optic nerve.

Introduction

The stimulus-evoked compound action potential (CAP) recorded from the rodent optic nerve is a long standing model system in the investigation of white matter injury resulting from anoxia (Stys et al., 1991b) and/or glucopenia (Brown et al., 2001a, Micu et al., 2006), with elucidation of injury mechanisms hopefully leading to clinically relevant neuroprotective therapies. In addition the rodent optic nerve has been used to study CNS regeneration (Cho et al., 2005, Sugioka et al., 1995), the role of myelin in multiple sclerosis (Waxman et al., 2004) and the role of auxiliary subunits in determining sodium channel function (Chen et al., 2002, Chen et al., 2004). The rodent optic nerve, cranial nerve II, is a purely white matter structure (Ransom et al., 1997) devoid of neuronal cell bodies and synapses. It is composed of myelinated axons, astrocytes and oligodendrocytes, and morphological studies using electron microscopy techniques have revealed that the optic nerve from an adult CD1 mouse contains about 25,000 axons, all of which are myelinated, with axons diameters varying from about 0.1 to 2.5 μm (Allen et al., 2006). Studies using fructose as the sole energy substrate strongly suggest that the axons contributing to the 1st CAP peak are the largest in size, with the smaller diameter axons contributing to the 2nd and 3rd CAP peaks (Allen et al., 2006). The initial rat model has been complemented by the mouse to ensure compatibility with future transgenic and spontaneous mutant models, and the decreased diffusion distances offered in the mouse is a key factor in in vitro metabolic studies where the blood borne delivery of nutrients is circumvented.

The evoked CAP resulting from a supramaximal stimulus has a stereotyped polyphasic profile consisting of three separate, but overlapping peaks. Conventional analysis has tended to use the total CAP area as the index of nerve function (Stys et al., 1991a). The rodent optic nerve is a model of choice in white matter injury since the ratio of CAP area pre- and post-insult indicates the degree of injury incurred by the nerve, and also offers a benchmark against which neuroprotective strategies can be evaluated (Brown and Ransom, 2002, Brown et al., 2001a, Brown et al., 2001b, Fern et al., 1993, Fern et al., 1995, Stys et al., 1990, Stys et al., 1992). In the majority of experiments a supramaximal stimulus is imposed on the nerve to evoke all available axons. Since action potentials are all or nothing a decrease in the area of the CAP is assumed to reflect a decrease in the number of axons contributing to the CAP (Stys et al., 1991a). However, recent results from our laboratory have indicated that the individual peaks exhibit differential sensitivities in response to energy substrate availability (Meakin et al., 2007). This is an important point, as it illustrates that neighbouring axons within the same tissue cannot all be assumed to respond in identical ways to a given insult.

In this present paper we demonstrate that resolving the CAP into its constituent peaks reveals heterogeneous metabolic properties of axon-subpopulations in mouse optic nerve, that preclude the assumption of axon homogeneity, at least with regard to energy substrate utilisation. In addition we demonstrate that complex CAP profiles comprising overlapping peaks of both positive and negative polarity, such as that recorded from the rumpshaker mouse, can be resolved satisfactorily into their constituent peaks. In conclusion we demonstrate that resolving CAPs into their constituent peaks can be a powerful analytic tool for revealing heterogeneous properties of axon-subpopulations underlying the CAP of rodent optic nerve.

Section snippets

Materials and methods

All procedures were performed in accordance with the Animals (Scientific Procedures) Act, 1986, under appropriate authority of project and personal licences. The rumpshaker (Plpjp-rsh) mutant mouse (Ile186Thr) was maintained on a C3H/101 background and has been described in detail previously (Griffiths et al., 1990).

Control CAPs

The CAP recorded from the optic nerve of an adult CD1 mouse has a typical polyphasic profile, comprising three peaks, as illustrated previously for both mouse (Brown et al., 2003) and rat (Stys et al., 1991a). The polyphasic nature reflects the heterogeneous velocity of propagation of the various axons. The large stimulus artefact is followed by the 1st CAP peak, which is intermediate in amplitude, and denotes axons with the fastest conduction velocity (Fig. 1C). The 2nd CAP peak exhibits the

Discussion

Since the introduction of the rodent optic nerve as a model for white matter injury and the subsequent detailed description of the electrical properties of the stimulus-evoked CAP (Stys et al., 1991a), this model system has been used to investigate white matter injury as a result of anoxia and/or aglycaemia. Such studies have without exception used the total CAP area as a measure of nerve conduction, the tacit assumption being that all axons in the nerve are a homogeneous population. Analysis

Grant support

Supported by a Grant from the Institute of Neuroscience, University of Nottingham. MMcL supported by the Wellcome Trust.

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